This application relates to lymphokines. In particular, it relates to lymphotoxin and derivatives thereof.
Lymphotoxin was first identified as a biological factor with anticellular activity on neoplastic cell lines. A substance identified as lymphotoxin and obtained from mitogen-stimulated lymphocytes has been demonstrated to have associated with it a spectrum of cytotoxic activities ranging from cytostasis of certain tumor cell lines to marked cytolysis of other transformed cells. On the other hand, lymphotoxin exhibits little or no anticellular activity on primary cell cultures and normal cell lines. This putative discriminating anticellular property of lymphotoxin led to in vivo studies which suggest that lymphotoxin may have a potent antitumor activity.
Purification and subsequent characterization of lymphotoxin proved difficult due to the small amount of activity secreted by primary lymphocyte cultures. The isolation and characteristics of lymphotoxin produced by the human lymphoblastoid cell line RPMI-1788 are described in Aggarwal et al., 1984, "J. Biol. Chem." 259(1): 686-691 and European patent application 100641 (published Feb. 15, 1984). This lymphotoxin is hereafter refered to as "lymphoblastoid" lymphotoxin.
Literature that should be studied in connection with this application includes J. Sawada, et al., 1976, "Jpn. J. Exp. Med." 46: 263-267; G. Granger, et al., 1978, "Cell. Immunol." 38: 388-402; J. Rundell, et al., 1981, "Immunopharmacology" 3:9-18; G. Granger, et al., 1982, "J. Lymphokine Res." 1: 45-49; N. Ruddle, et al., 1983, "Lymphokine Res." 2: 23-31; H. Ohnishi, et al., U.K. patent application No. 2,117,385; M. Mitsuhashi, et al., U.K. patent application No. 2,106,117; H. Enomoto, European patent application No. 87,087; B. Williamson, et al., 1983, "Proc. Natl. Acad. Sci. USA" 80: 5397-5401, and S. Wright et al., 1981, "J. Immunol." 126: 1516-1521.
It should be understood that lymphokine terminology is not uniform. At present, the names given to cell culture products are largely a function of the cells which elaborate the product and the performance of the products in biological assays. However, these products remain largely uncharacterized and their true identity will remain unknown in the absence of standard terminology based on clearly assayable distinguishing characteristics such as amino acid sequences or immune epitopes. Other names given to cytotoxic cell culture products include tumor necrosis factor, NK cell cytotoxic factor, hemorrhagic necrosis factor and macrophage cytotoxin or cytotoxic factor.
Lymphotoxin from RPMI-1788 has been isolated from culture supernatants as a putative aggregate having a molecular weight of about 60,000 Kd. This aggregate is resolved by purification into two molecular weight variants which differ in their amino terminal sequence. The larger variant, shown in FIG. 2a at amino acids 1-171 and having molecular weight of about 25,000 daltons, is distinguished by a leucyl amino terminal residue (hereafter "leucyl amino-terminal lymphotoxin"). The smaller 20,000 dalton variant also shown in FIG. 2a amino acids 24-171 contains a histidyl amino terminal residue (hereafter "histidyl amino-terminal lymphotoxin"). The literature contains reports of cytotoxic proteins found in supernatants from lymphocytes or lymphoblastoid cell lines which vary widely in molecular weight, from 12,000 to more than 200,000. It would be desirable to manufacture lymphotoxin having a substantially uniform molecular weight and uniform amino terminal amino acid sequence.
The lymphotoxin (or substances identified as lymphotoxin) obtained heretofore from lymphocyte culture are present in low concentrations, on the order of 0.05-2.times.10.sup.6 units/l in supernatants of RPMI-1788 cells or peripheral blood lymphocytes. An economical method for producing uniform lymphotoxin is needed. Although the antitumor effects and apparent therapeutic value of lymphotoxin have been reported in the literature since at least 1976, lymphotoxin has not been studied in extensive clinical protocols or commercialized due to the small quantities and heterogenous nature of lymphotoxin produced in lymphocyte culture.
The characterization and purification of lymphotoxin would be facilitated by antibody raised against the lymphotoxin active or receptor binding site or an adjacent region which neutralizes the cytotoxic activity of lymphotoxin. The need for this antibody has been manifest for some time in the field of this application in view of the poorly characterized nature of cytotoxic lymph cell products and the need to purify and assay lymphotoxin, but as far as is presently known no publication of such an antibody is extant.